Microbial Genetics Transformation PDF

Transformation Recall DNA exchange methods: Conjugation, transduction, and transformation. Conjugation: Plasmids or self-transmissible DNA transfer between bacterial cells. Transduction: Phages carry DNA from one bacterium to another. Transformation: Cells take up free DNA from their e...

Transformation Recall DNA exchange methods: Conjugation, transduction, and transformation. Conjugation: Plasmids or self-transmissible DNA transfer between bacterial cells. Transduction: Phages carry DNA from one bacterium to another. Transformation: Cells take up free DNA from their environment, key in molecular genetics. Natural Transformation Natural transformation: Some bacteria can naturally take up DNA without special treatments. Competence: Bacteria need to be in a specific state to take up DNA; those that can naturally reach this state are called naturally competent.  Discovery of Transformation Fred Griffith's experiment (1928): Found that nonpathogenic S. pneumoniae could be transformed into pathogenic forms by mixing dead pathogenic cells with live nonpathogenic cells. Oswald Avery's work: In the 1940s, Avery and collaborators identified this "transforming principle" as DNA, demonstrating it as the hereditary material. Transformation in Gram Positive Bacteria B. subtilis and S. pneumonia are well studied gram +ve bacteria that can take up DNA from any organism Includes pseudopilus formed by ComG proteins, which interact with DNA for uptake; NucA endonuclease cleaves DNA for transport. Transformation in Gram Positive Bacteria Com genes: Involved in transformation; mutations result in competence defects. Organization of com genes: In B. subtilis, these genes form operons; comA and comK regulate competence, while comE, comF, and comG form part of the DNA transport machinery. comEA gene: Encodes protein binding extracellular double-stranded DNA.  Transformation in Gram Positive Bacteria DNA processing: One strand is degraded, the other is transported through ComEC channel with ComFA as an ATP-dependent translocase. EndA nuclease: Generates single-stranded DNA in S. pneumoniae; corresponding activity in B. subtilis is unknown. DNA transport: Single-stranded DNA moves into the cell at 80-100 nucleotides per second, with 3′-to-5′ polarity in S. pneumoniae. Transformation in Gram Negative Bacteria Utilize type II secretion systems and type IV pili, similar to pseudopilus systems in gram-positive bacteria. Pseudopilus function: May grow to push proteins out (type II secretion) or retract to pull DNA in (transformation). Transformasomes: capture and process double- stranded DNA for transport through the inner membrane. DNA Processing after Uptake Protection of single-stranded DNA (ssDNA): Covered with single-stranded-DNA-binding protein (SSB) to prevent degradation. RecA protein: Binds to ssDNA and mediates homologous recombination with the chromosome. Homologous recombination: Occurs when the transformed DNA is similar to the cell's resident DNA; involves strand displacement. DNA Processing after Uptake Recombinant types: Appear if donor and recipient DNA sequences differ slightly. DNA integration: Single-stranded DNA segments of about 8.5 to 12 kb are incorporated into the recipient chromosome within minutes. Plasmid Transformation and Phage Transfection of Naturally Competent Bacteria Chromosomal DNA transformation: Efficient in naturally competent bacterial cells of the same species. Plasmid/phage DNA limitations: Cannot efficiently enter naturally competent cells because they need to be double- stranded and capable of recyclizing. Competence Pheromones B. subtilis signals high cell density through small peptides called competence pheromones, which are released as bacteria multiply. Quorum sensing: Cells become competent only in high concentrations of pheromones, ensuring DNA uptake happens only when other B. subtilis cells are nearby, utilizing a phenomenon called quorum sensing. Competence Pheromones Relationship between Competence, Sporulation, and Other Cellular States Dual processes: At the stationary phase, B. subtilis cells either become competent or start sporulation. Regulatory peptides: B. subtilis produces peptides (Phr) to regulate sporulation and competence indirectly by inhibiting Rap proteins, thus allowing ComA to activate transcription. Role of Natural Transformation Nutrition Repair Recombination Importance of Natural Transformation for Forward and Reverse Genetics Natural transformation is widely used in molecular genetics for mapping genetic markers and reintroducing manipulated DNA into cells. Congression: Multiple DNA-binding sites on a single cell enable the import of multiple DNA segments, impacting genetic mapping and plasmid DNA transformation. Artificially Induced Competence Chemical Induction-Generally inefficient, requiring selective plating conditions and the use of selectable genes in the DNA Electroporation introduces DNA into bacterial cells by exposing them to a strong electric field, creating temporary hydrophilic pores in the membrane. Works with most types of cells, including most bacteria, making it more versatile than other species-specific methods Artificially Induced Competence Protoplast transformation involves disrupting the bacterial cell wall, creating osmotically sensitive protoplasts that require high- osmolarity solutions for stability. most efficient with circular plasmid DNAs that can establish independent replication within the transformants. Thank You Fully Editable Shapes  Fully Editable Icon Sets: A You can Resize without losing quality You can Change Fill Color & Line Color FREE PPT TEMPLATES www.allppt.com  Fully Editable Icon Sets: B You can Resize without losing quality You can Change Fill Color & Line Color FREE PPT TEMPLATES www.allppt.com  Fully Editable Icon Sets: C You can Resize without losing quality You can Change Fill Color & Line Color FREE PPT TEMPLATES www.allppt.com